Protein Binders and Their Applications in Developmental Biology Stefan Harmansa* and Markus Affolter‡
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© 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev148874. doi:10.1242/dev.148874 PRIMER Protein binders and their applications in developmental biology Stefan Harmansa* and Markus Affolter‡ ABSTRACT Gettemans, 2017; Helma et al., 2015; Plückthun, 2015; Sha et al., Developmental biology research would benefit greatly from tools that 2017). These genetically encodable binders, which are based on enable protein function to be regulated, both systematically and in a various protein scaffolds, can be used to block or mask protein ‘ ’ precise spatial and temporal manner, in vivo. In recent years, function. Furthermore, such protein binders can be functionalized functionalized protein binders have emerged as versatile tools that by fusing them to various effector domains with the ultimate goal of can be used to target and manipulate proteins. Such protein binders directly visualizing or regulating the function and interaction of can be based on various scaffolds, such as nanobodies, designed target proteins in living cells or organisms. ankyrin repeat proteins (DARPins) and monobodies, and can be used In the past few years, protein binders have started to be used in to block or perturb protein function in living cells. In this Primer, we developmental biology, and a multitude of novel protein binder- provide an overview of the protein binders that are currently available based tools has been reported. In this Primer, we provide an and highlight recent progress made in applying protein binder-based overview of these tools and discuss the possible applications of tools in developmental and synthetic biology. protein binders in developmental biology research. Furthermore, we highlight the potential and advantages of adding protein binder- KEY WORDS: Protein binder, Nanobody, DARPin, Monobody, based tools to the methodological repertoire of developmental Protein degradation, GFP biologists. Introduction Types of protein binders and their generation Forward and reverse genetic approaches have been key to expanding For many years, antibodies have been the tools of choice for our knowledge of gene function during development (see Housden recognizing a specific protein of interest (POI), and they are still the et al., 2017 and references therein). However, as more and more most widely used reagents for many applications. However, owing to proteins and pathways are becoming associated with developmental their large size and poor folding and stability properties in the processes, and as increasingly complex networks of regulatory cytoplasm of cells, antibodies are most often used in the extracellular interactions emerge (see for example Manning and Toker, 2017), milieu or in fixed tissues, and have proven to be less useful in the the need to study protein function in detail at tissue and single-cell context of a live cell. Over the last decade, however, several classes of scales in living organisms has become greater than ever. This smaller protein-binding scaffolds have been used to generate protein- represents a considerable challenge, in particular when studying specific binders that function when expressed in cells. proteins that regulate general cellular processes or have broad The different classes of peptide-based binders that currently exist expression patterns. The availability of tools that allow protein can be divided into two broad families: (1) those that are based on or function to be regulated more precisely and acutely in a spatial and derived from immunoglobulins, i.e. antibodies and derivatives temporal manner would be extremely helpful and could provide thereof, such as single-chain variable fragments (scFvs) and unprecedented insights into complex developmental processes. nanobodies; and (2) those that are based on non-immunoglobulin, Over the years, several methods have been developed to natural or designed protein scaffolds, such as designed ankyrin manipulate proteins directly in vivo. These include degradation- repeat proteins (DARPins), monobodies, affibodies, anticalins and inducing applications (Banaszynski et al., 2006; Bonger et al., 2011; others (Boxes 1-3, Fig. 1). In the past decade, tremendous progress Chung et al., 2015; Natsume et al., 2016), protein cleavage using has been made in refining these scaffolds for better stability, higher tobacco etch virus (TEV) protease (Harder et al., 2008; Pauli et al., affinity and easier handling (Goldman et al., 2017; Schilling et al., 2008), the ‘anchor-away’ approach (Haruki et al., 2008), the 2014; Schmidt et al., 2016), and libraries of increasing complexity ‘knocksideways’ technique (Robinson et al., 2010) and various have been generated and characterized (Moutel et al., 2016; Tiede dimerization tools that allow protein functions to be assembled in an et al., 2017; Yan et al., 2014). inducible manner (Renicke et al., 2013; van Bergeijk et al., 2015; With the exception of immunoglobulin-based binders, which can Wu et al., 2009), to mention just a few. However, an additional, also be obtained upon immunization (Greenfield, 2014), protein more systematic approach to manipulation of protein function has binders are generally selected using in vitro display techniques recently emerged. This approach utilizes protein binders, which are (Boder and Wittrup, 1997; Samuelson et al., 2002; Zhao et al., small, protein-based affinity reagents that can selectively recognize 2009), with phage display being the most commonly used and bind to a target protein and that are increasingly being used to (Bradbury et al., 2011; Breitling et al., 1991; Romao et al., 2016; study protein function in living cells and organisms (Beghein and Kuhn et al., 2016). Although these techniques use different biological systems, their general principles are shared; they allow large peptide libraries to be screened, the respective protein is Growth and Development, Biozentrum, University of Basel, 4056 Basel, Switzerland. *Present address: IBDM, UMR 7288, Aix-Marseille Universitéand CNRS, 13009 coupled to its encoding DNA sequence, and multiple cycles of Marseille, France. selection can be used to increase binding specificity (Fig. 2). The isolation of protein binders can also be performed commercially and ‡Author for correspondence ([email protected]) on large platforms, and many institutes have established core S.H., 0000-0001-6668-7608; M.A., 0000-0002-5171-0016 facilities to isolate and characterize specific protein binders, making DEVELOPMENT 1 PRIMER Development (2018) 145, dev148874. doi:10.1242/dev.148874 characterization of a novel binder. The latter then has to be Box 1. scFv scaffolds validated with regard to its specificity, efficacy and off-target Conventional antibodies (immunoglobulins, IgGs; see Fig. 1A) have effects, a task that is not always trivial and has to be carefully designed been used extensively in basic research and are indispensable tools for and executed. protein detection. However, conventional IgGs are unsuitable for The second step isto functionalize the binder such that it modifies or intracellular expression for various reasons; the reducing nature of the regulates the target protein in a desired manner. This represents one of intracellular environment hampers disulphide bond formation and thus proper antibody folding, and whole IgG antibodies have a complex the most exciting aspects of protein binders, as there are innumerable structure and a high atomic mass (∼150 kDa). These drawbacks are possibilities for such functionalization. Over the past few years, the partially overcome by connecting the VH and VL domain with a peptide availabilityof awide range of high-affinity protein binders has resulted linker, forming a so-called single-chain variable fragment (scFv; Bird et al., in the development of a versatile repertoire of novel protein binder- 1988; Huston et al., 1988; see Fig. 1B) that retains antigen-binding based techniques and tools (reviewed by Bieli et al., 2016; Böldicke, ∼ capacity. scFvs are relatively small ( 28 kDa) and consist of a single 2017; Helma et al., 2015; Kaiser et al., 2014; Marschall et al., 2015; domain that can be expressed in various host systems, such as bacteria, yeast or higher animals. These advantages make scFvs attractive tools in Plückthun, 2015; Sha et al., 2017). Many of these tools have been medical applications and in biotechnology (Lyon and Stasevich, 2017; validated using binders that recognize specific fluorescent proteins. In Monnier et al., 2013) but their use as intracellular protein binders particular, several binders against green fluorescent protein (GFP) (intrabodies) is restricted because scFvs typically contain two highly have been isolated and characterized (Fridy et al., 2014; Kirchhofer conserved intra-domain disulphide bonds (Williams and Barclay, 1988). et al., 2010; Kubala et al., 2010; Rothbauer et al., 2006; Saerens et al., These bonds influence scFv stability and function (Glockshuber et al., 2005; Brauchle et al., 2014). In addition, several studies have used 1992; Proba et al., 1997), and so only intrinsically stable scFvs fold correctly within a cell and can be utilized as functional intrabodies (Worn binders that recognize specific small tags, such as the 19 amino acid and Pluckthun, 1998). Owing to these limitations, relatively few scFv- (aa) peptide derived from GCN4 used in SunTag (discussed below) based binders have been used to date in developmental biology. (Tanenbaum et al., 2014; Wörn et al., 2000). Nonetheless, using protein engineering (Tanha et al., 2006)